The free oxygen radical, nitric oxide (NO), is elevated in a variety of malignant tumors in vitro and in vivo. Three isoenzymes of nitric oxide synthase (NOS) are responsible for the synthesis of NO from arginine in the organism: constitutive neuronal NOS (nNOS or NOS 1), identified mainly in neurons; inducible NOS (iNOS or NOS 2), found in macrophages and other cells; and constitutive endothelial cell NOS (eNOS, ecNOS, or NOS 3), found predominantly in endothelial cells. Elevated levels of NO have been associated with increased tumor aggressiveness in many forms of cancer. Increased NO levels have been found in most breast cancers examined and are associated with accelerarion of angiogenesis, abrogation of apoptosis, and tumor invasion. In the present R03 application, we propose to elucidate the role of NO generation in the invasive characteristics of breast cancer cells. We hypothesize that increased levels of NO (produced by increased NOS activity) are, indeed, associated with enhanced tumor aggressiveness. To directly examine the role of NOS in cells, we will study murine mammary cancers induced by the carcinogen 7-12- dimethybenz(a)anthracene (DMBA) in iNOS and eNOS knockout mice. These models will enable us to identify the type of NOS that is most associated with tumor aggressiveness. We also will test our hypothesis by chemicall altering the amount of NO in the cellular environment using NOS inhibitors. Inhibition of NOS activity by NG-nitro-L- arginine methyl ester (L-NAME) or by NG-methyl-L-arginine (NMMA) should reduce tumor growth and metastases if NO is a significant positive factor in these processes. Finally, we anticipate that addition of exogenous NO that does not require NOS for its production (e.g., administration of sodium nitroprusside or NOC-12 [1hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-l-triazine] will increase tumor invasiveness. We anticipate that the results obtained from these preliminary studies will provide us with sufficient data to lay the foundation for a subsequent R01 application exploring the mechanisms involved in the actions of NO and NOS in breast cancer, with ultimate translation to human disease, potentially in terms of both biological markers and them therapeutic interventions.